Fronts divide diazotroph communities in the Southern Indian Ocean

Abstract Dinitrogen (N2) fixation represents a key source of reactive nitrogen in marine ecosystems. While the process has been rather well-explored in low latitudes of the Atlantic and Pacific Oceans, other higher latitude regions and particularly the Indian Ocean have been chronically overlooked. Here, we characterize N2 fixation and diazotroph community composition across nutrient and trace metals gradients spanning the multifrontal system separating the oligotrophic waters of the Indian Ocean subtropical gyre from the high nutrient low chlorophyll waters of the Southern Ocean. We found a sharp contrasting distribution of diazotroph groups across the frontal system. Notably, cyanobacterial diazotrophs dominated north of fronts, driving high N2 fixation rates (up to 13.96 nmol N l−1 d−1) with notable peaks near the South African coast. South of the fronts non-cyanobacterial diazotrophs prevailed without significant N2 fixation activity being detected. Our results provide new crucial insights into high latitude diazotrophy in the Indian Ocean, which should contribute to improved climate model parameterization and enhanced constraints on global net primary productivity projections.


Introduction
Pr okaryotes called "diazotr ophs" fix dinitr ogen (N 2 ) into ammonium, sustaining primary productivity and carbon export in the ocean (Karl et al. 1997, Zehr and Capone 2020, Bonnet et al. 2023 ).Although c y anobacteria ar e conv entionall y r egarded as the primary contributors to marine N 2 fixation, non-c y anobacterial diazotr ophs (NCDs) ar e ubiquitousl y distributed in marine ecosystems (Turk-Kubo et al. 2022 ).The activity of diazotrophs is importantl y contr olled by temper atur e (Sohm et al. 2011 ), as well as the availability of phosphorus and iron (Fe) (Mills et al. 2004 ).N 2 fixation can be inhibited by r eactiv e nitr ogen compounds suc h as ammonium or nitr ate (LaRoc he and Breitbarth 2005 ).Under such constraints, N 2 fixation has long been assumed to be restricted to w arm lo w-latitude nitr ate-poor open ocean r egions, pr ovided sufficient phosphorus and Fe are available (Zehr and Capone 2020 ).Ho w e v er, r ecent studies r e ported significant N 2 fixation acti vity in cold an nutrient-rich regions, including temperate (Raes et al. 2020 ), and polar waters (von Friesen and Riemann 2020 ).
The Southern Indian Ocean (SIO) region comprises the Indian Ocean (IO) subtropical gyre and the Indian sector of the Southern Ocean.The IO subtropical gyre is characterized by low dissolved nitr ogen-to-phosphorus (N:P) r atios ( ∼4:1) in the photic zone (Baer et al. 2019 ), conditions belie v ed to favour diazotr ophy (Kna pp 2012 ).Ho w e v er, low F e a v ailability in the IO subtr opical gyr e may hinder N 2 fixation and intensify nitrogen limitation (Grand et al. 2015 ).Recurring massive blooms of Trichodesmium , a prominent diazotrophic c y anobacterium, and frequent occurrence of diatomdiazotroph associations are observed at the southern tip of Madagascar (Poulton et al. 2009 ).These observations point to w ar ds an important contribution of diazotrophs to nitrogen fluxes in the IO's subtropical gyre (Poulton et al. 2009, Metzl et al. 2022 ).
The IO subtropical gyre is separated from the Indian sector of the Southern Ocean by a complex succession of quasi-zonal fr ontal structur es, including the subtr opical fr ont (STF), subantar ctic F r ont (SAF), and polar fr ont (PF) (Kostianoy et al. 2004, Chapman et al. 2020 ).While fronts are usually considered as barriers , recent studies ha ve shown that front-associated filaments facilitate sub/mesoscale transfer of seawater properties and communities between the Southern Ocean and the IO subtropical gyre across the fronts (Read et al. 2000, Hörstmann et al. 2021 ).Fronts can enhance vertical fluxes of nutrients (D'Asaro et al. 2011 ), resulting in high primary productivity, and typically distinct microbial communities as compared to the surrounding water masses (Baltar et al. 2016, Hörstmann et al. 2021 ).While some studies ha ve in vestigated diazotrophy across frontal systems (Fong et al. 2008, Benavides et al. 2011, 2021, Riou et al. 2016, Shiozaki et al. 2018, Jiang et al. 2019 ), their role in structuring diazotroph comm unities and contr olling N 2 fixation inputs is lar gel y unknown (Benavides and Robidart 2020 ).
The Southern Ocean is rich in macronutrients but low in productivity due to the lack of F e , for which it is known as a "high nutrient low c hlor ophyll" (HNLC) r egion (Venables and Moor e 2010 ).Diazotr ophs r el y heavil y on F e , a vital component of the nitrogenase enzyme (Berman-Frank et al. 2001 ).The lack of Fe together with the cold-water conditions and high nitrate concentrations of the Southern Ocean IO sector are thus expected to suppress diazotrophy.Ho w ever, a range of processes provides intermittent sources of Fe in this region, including atmospheric deposition, fr ontal jet inter actions with bathymetry (Blain et al. 2007, Klocker 2018 ), and Fe from hydrothermal origin (Ardyna et al. 2019, Schine et al. 2021 ), known to prompt phytoplankton blooms and possibl y diazotr ophic acti vity as well.In ad dition to F e , bioactiv e tr ace metals such as manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn) are essential enzyme cofactors, controlling the growth of primary producers in the open ocean (Saito et al. 2008, Sunda 2012, Balaguer et al. 2023 ).Ho w ever, the effects of trace metals , excluding F e , on diazotrophs are not well understood.
An intercomparison of the latest generation of Earth system models shows that the IO is one of the main ocean basins adding uncertainty to global net primary productivity projections (Tagliabue et al. 2021 ).Hence, increasing N 2 fixation measurements and its controlling mechanisms in the IO is crucial for improving the predictability of net primary productivity and understanding the ocean's future role as a climate change regulator.Seeking to impr ov e the cov er a ge of diazotrophy studies in the SIO, here we investigate N 2 fixation rates and the diazotroph community composition and abundance along a transect spanning from the oligotr ophic IO subtr opical gyr e to the HNLC waters of the Southern Ocean.T his study pro vides v aluable data to impr ov e our understanding of high latitude diazotrophy and nitrogen budgets in the IO.

Sample collection and environmental parameters
This study was conducted from 13 January to 4 March 2021 (austral summer) onboard the R/V Marion Dufresne II as part of the Fr enc h SWINGS (GEOTRACES GS02) cruise (doi: 10.17600/18001925) (Fig. 1 ).Surface seawater was collected at eight stations using Niskin bottles mounted on a conductivitytemper atur e-depth (CTD) r osette pac ka ge and at 43 other stations using the underway sampling system (Fig. 1 ).Underway sampling consisted of a water intake at the base of the ship hull through the moonpool of the ship and a PTFE pump and tubing minimizing trace metal contaminations.Sea surface temper atur e (SST) and salinity data were retrieved from the CTD sensors.Chlorophyll a concentrations were retrieved from Aqua MODIS Satellite data (from 13 January to 4 March 2021, L3M 4 km product).Samples for dissolved inorganic nutrients and trace metals were collected from CTD casts using a trace metal clean polyurethane powder-coated aluminum frame rosette equipped with 24 12-l, externall y closing, Teflon-lined, GO-FLO bottles (Gener al Ocean-ics) and attached to a Kevlar ® line, according to the GEOTRACES guidelines (Cutter et al. 2017 ).Seawater samples for dissolved inor ganic nutrients anal ysis (nitr ate , nitrite , phosphate , and silicic acid) wer e pr efilter ed thr ough 0.45-μm acetate cellulose filters, poisoned with HgCl 2 (4 g l −1 ) and stored in the dark at room temper atur e until anal ysis using a Skalar segmented flow autoanalyzer (Blain et al. 2015 ).Detection limits for the nitrate , nitrite , phosphate, and silicic acid were 0.15 μmol l −1 , 0.01 μmol l −1 , 0.02 μmol l −1 , and 0.07 μmol l −1 , r espectiv el y.
Dissolved nutrients and trace metal concentration data are used here for statistical purposes, while a full description of their variability during the SWINGS cruise will be provided in dedicated papers (e.g.Baudet et al., submitted).

N 2 fixation and primary production rates
Samples for the measurement of primary production and N 2 fixation rates were collected from the underway outlet as a set of four 2.3-l acid-washed polycarbonate bottles .T hree bottles were spiked with 2.3 ml of a 13 C-labelled bicarbonate solution (NaH 13 CO 2 ; > 98%, Sigma Aldrich, 10 atom% final 13 C abundance) and 2.3 ml of 15 N 2 ( 15 N isotopic abundance of 99.7%, Eurisotop, Saclay, France) using the bubble method to maximize the final 15 N isotopic abundance and impr ov e the detection limit.Bottles were inverted at least 60 times before incubation to ensur e r a pid isotopic equilibrium.The fourth bottle was left unamended and used as a control.All the bottles were incubated for 24 h in an on-deck incubator with circulating surface water and a blue filter r epr oducing the light at the sampling depth.Temper atur e c hanges between the start and the end of the incubation were limited to 1.2 • C on av er a ge.Following incubations, 12 ml of water were syphoned from the bottles in Exetainer tubes and poisoned with HgCl 2 for 15 N 2 isotopic abundance analyses .T he remaining bottle content was filtered on combusted (450 • C, 4 h) 25-mm diameter glass fibre filters (Whatman, London, UK).Filters were stored at −20 • C before being dried for downstream analysis (24 h, 60 • C).The 15 N 2 isotopic abundance in water was measured within 6 months using a membrane inlet mass spectrometer according to Kana et al. ( 1994 ).The particulate carbon and nitrogen isotopic enrichment ( 13 C and 15 N) was measured on an elemental analyzer coupled to an isotope ratio mass spectrometer (Deltaplus , T hermo Finnigan).N 2 fixation rates were calculated according to Montoya et al. ( 1996 ).Minimum quantifiable rates were also calculated based on err or pr opa gation of the r eplicates as pr oposed by White et al. ( 2020 ).
Single-cell N 2 fixation r ates wer e measur ed by nanoscale secondary ion mass spectrometry (nanoSIMS) as described in Bonnet et al. ( 2016 ) at station 857 (north of the fronts) targeting Crocosphaera -like cells and Trichodesmium , and at four stations south of the fronts (stations 872, 876, 887, and 893) targeting putati ve NCDs.Putati ve NCDs were not measured north of the fronts (where c y anobacterial N 2 fixation rates were expected to be high), to avoid false positives due to the transfer of enriched 15 N biomass from c y anobacterial diazotrophs to heterotrophic bacteria (Bonnet et al. 2016 ).Briefly, at station 857 cells were deposited on a polycarbonate filter (0.2 μm pore size) following 15 N 2 incubation.Trichodesmium filament and Crocosphaera -like cells were mapped by epifluor escence befor e nanoSIMS anal yses.At stations 872, 876, 887, and 893, incubated cells were laid on a polycarbonate filter (0.2 μm pore size) and resuspended in 4.5 ml fr eshl y pr oduced filtered (0.2 μm pore size) seawater, fixed with 1% paraformaldehyde and flash frozen in liquid nitrogen.Cells were then stored at −80 • C. Back in the laboratory, samples w ere thaw ed and cells were labelled with SYBR green I DNA dye (Molecular Probes, final concentr ation 0.05%).Heter otr ophic bacteria wer e gated according to Marie et al. ( 1999 ) and sorted using a BD FACSAria™ Fusion cell sorter.Sorted heter otr ophic bacteria were directly deposited on a polycarbonate membrane on the sorter outlet as described by Bonnet et al. ( 2016 ).In total, 50 c y anobacterial diazotrophs w ere analyzed, and 2,535 heterotrophic bacteria.Incubation time was 24 h for station 857 and between 48 and 168 h, long enough to increase the likelihood of detecting positiv el y enric hed NCDs.Cells wer e consider ed as significantl y 15 N enric hed when the number of 15 N ions counted exceeded the number of 15 N ions assuming a natural abundance (0.366 atom%) plus three times the standard deviation of the Poisson distribution modelled for each cell (see Berthelot et al .(2019) for more details).

DN A sampling, extr action, diazotroph abundance, and nifH/nifD gene sequencing
Nucleic acids were sampled from 51 stations, 2-l seawater samples filter ed thr ough 0.2-μm pol ycarbonate filters, and stor ed at −80 • C until analyzed.DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, CA, USA) and quantified by the Picogreen method using a VarioSkan spectr ofluor ometer (Thermo Fisher Scientific , Massachusetts , USA).For DN A extraction, w e used additional modifications including freeze-thaw with liquid nitrogen, bead beating, and Proteinase-K steps before the kit purification and elution to 70 μl in RNase-free water, as previously described (Moisander et al. 2008 ).The abundance of nif H genes was quantified using TaqMan-specific quantitative PCR (qPCR) assays targeting six diazotroph groups including Trichodesmium , UCYN-A1, UCYN-A2, UCYN-B, Gamma-A, and Gamma-4, using published primer-probe sets (see Supplemental files ).
The nif H gene was amplified using degenerated oligonucleotide primers ( nif H1-2-3-4) (Zehr and McReynolds 1989 ), to produce a final amplicon length of 359 base pairs (bp).In diazotroph community studies, nifH is frequently emplo y ed as a biomarker gene to identify diazotrophs and their community composition (Frank et al. 2016, Gaby et al. 2018 ).Howe v er, to perform N 2 fixation diazotr ophs r equir e a minim um set of genes ( nif HDKENB; Dos Santos et al. 2012 ).Up to 20% of nif H-harbouring genomes sho w ed the absence of the genes nif D and nif K, and this genomic configuration is referred to as pseudo nif H (Mise et al. 2021 ).Pseudo nif H has been found in various common diazotroph groups, including Clostridia and methanogens (Mise et al. 2021 ).A recent stud y ad vocated rethinking using nif H as the primary biomarker for N 2 fixing microbes and proposed to consider the nif D and nif K genes instead (Mise et al. 2021 ).Here, we used a dual gene amplicon a ppr oac h ( nif H and nif D genes) to comprehensiv el y explor e diazotr oph comm unity composition.To amplify nif D genes, nested PCRs were performed to produce a final amplicon length of 512 bp (McRose et al. 2017 ).Amplicons were checked using a 1.2% a gar ose gel stained with ethidium bromide, and ima ges wer e ca ptur ed using a UV tr ansilluminator.nif H and nif D PCR fr a gments wer e purified by using the MP Biomedicals Geneclean Turbo Kit (Fisher Scientific , Massachusetts , USA).Gel-purified PCR pr oducts wer e shipped to Macrogen, Inc. (Amsterdam, Netherlands) for the clone library preparation, adapter ligation, and Illumina MiSeq 2 × 300 bp paired-end sequencing.

Bioinformatics
Dem ultiplexed r aw Illumina sequence r eads wer e r eceiv ed fr om the sequencing facility and primer and adapter sequences remov ed.Sequences wer e filter ed, trimmed, and pr ocessed using the D AD A2 (V.1.16) pipeline with default settings (Callahan et al. 2016 ).After r e vie wing the quality pr ofiles, sequences with a quality score > 30% were k e pt for downstream processing.Upon concatenating all the pr ocessed mer ged r eads fr om the forw ar d and r e v erse sequences, onl y sequences exceeding a minimum length of 300 bp were retained for subsequent downstream analyses.Chimer as wer e r emov ed to get rid of the artefact sequences created b y tw o or more biological sequences that were wrongly linked together.Post c himer a r emov al, amplicon sequence variants (ASVs) with a length greater than 300 bp are selected for further analysis, including ASV table generation and taxonomic profiling.To exclude the potential non-nif H reads, we applied the specific following segments of the NifMAP pipeline v.1.0(Angel et al. 2018 ).(a) The filtration was conducted utilizing the Hidden-Markov Model (HMM) "hmm_nuc_1160_ nif H.hmm" through the application of hmmsearch in HMMER version 3.1b2 ( http:// hmmer.org/).(b) Amino acid translation, adjusting for potential frameshifts, was done using Framebot (Wang et al. 2013 ) against a nif H reference set.(c) Detection of nif H homologs ( bc hX , c hlL , bc hL , and parA ) w as done emplo ying the HMM "nif H_c hlL_bc hX.hmm" thr ough the utilization of hmmscan in HMMER.(d) Filtered ASVs were annotated into clusters using the CART model (Frank et al. 2016 ).ASVs that successfully underwent the NifMAP pipeline were retained for further downstream analysis .T he taxonomy of the nif H ASVs was assigned by the nif H database v2.0.5 (Moynihan and Reeder 2023 ).
We used the nifD database de v eloped by Furbo Reeder et al. ( 2023 ) (doi:10.5281/zenodo.10124357).For the de v elopment of the nif D database, ARBitrator (Heller et al. 2014 ) (version 14 April 2022) were used for retrieval of nif D sequences from GenBank matching a given set of reference sequences.Reference sequences were verified as true nif D using CD-search tool (CD-HIT) (Lu et al. 2020 ) to c hec k if they contained the conserved protein domain (cd01976).Settings for Arbitrator were as follows: quality and superiority thr esholds wer e set to 8.1 and 0.1, as in Heller et al. ( 2014 ).As a model for conserved domains (cd) models, cd01976 was used as a positive indicator while cd01967 was used as an indicator for an uninformative domain.nif D sequences were stored as EMBL r ecords, whic h wer e imported into ARB (Ludwig et al. 2004 ) to create a nif D ARB database.Finally, the nif D ARB database was exported in XML format.Sequence ID, taxa, fasta sequence, and accession number, wer e extr acted using Moynihan and Reeder ( 2023 ) R script.In total, we obtained 2438 nif D sequences.ASVs generated by nif D sequencing were assigned by our ne wl y formed nif D database nifD_DB_v1.1.0( https:// github.com/OceanBridges/ nifDdada2 ).
The top 100 ASVs from nif H and nif D were queried against the NCBI nucleotide databases to find related reference sequences.Sequences were aligned using the MUSCLE algorithm (Edgar 2004 ) in MEGA X (Kumar et al. 2018 ).To ascertain the optimal nucleotide substitution model and assess the need for gamma correction or estimation of invariable sites, the aligned nif H and nif D sequences underwent a model test using raxmlGUI 2.0 (Edler et al. 2021 ).Following the model selection process, determined by the Akaike Information Criterion, a maximum-likelihood phylogenetic tree was constructed with 1000 bootstr a p r eplicates using r axmlGUI 2.0 (Edler et al. 2021 ).The tr ees wer e subsequentl y visualized and annotated using iTOL ( https:// itol.embl.de/ ) (Letunic and Bork 2021 ).

Sta tistical anal yses
Since temper atur e is a major factor influencing the distributions of diazotrophs (Stal 2009, Sohm et al. 2011 ), we employed W ard' s method to cluster stations based on SST.This a ppr oac h aimed to enhance our understanding of diazotroph distribution patterns and their temper atur e pr efer ences.Data anal ysis was conducted using R (v4.2.0), and data visualization emplo y ed ggplot2 (v3.4.0) (Wic kham 2009 ).Dendr ogr am hier arc hical clustering with W ard' s method (Ward Jr. 1963 ) was applied to cluster the sampled stations based on the SST ( Fig. S1 , Supplemental files ).Pearson correlations were used to assess pairwise associations between inorganic nutrients , physical parameters , and tr ace metal concentr ations (collectiv el y defined here as "environmental variables"), N 2 fixation rates, and diazotroph nif H gene counts, using the rcorr function from the corrr package (v0.6.0)(Makowski et al. 2020 ).Differences in diazotr oph comm unity composition as assessed by nif H and nif D amplicon sequencing were investigated by nonmetric dimensional scaling analysis using the metaMDS from the vegan pac ka ge T he effect of en vir onmental v ariables on diazotr oph community composition and abundance was examined using redundancy anal ysis fr om the v egan pac ka ge (Oksanen et al. 2019 ).

Description of the study area and environmental settings
Str ong gr adients in SST, dissolv ed inor ganic nutrients, tr ace metals, and Chlor ophyll-a concentr ations wer e observ ed acr oss the fr onts separ ating the IO subtr opical gyr e and Southern Ocean waters.Among these variables, SST was the best discriminator to group stations along the cruise transect, as revealed by a dendrogr am anal ysis distinguishing thr ee distinct SST clusters including SST > 25 • C, SST between 10 • C and 25 • C, and SST < 10 • C ( Fig. S1 , Supplemental files ).
N 2 fixation and primary production rates N 2 fixation r ates r anged fr om below the detection limit to 13.96 nmol N l −1 d −1 north of the fronts, and from below the detection limit to 1.23 nmol N l −1 d −1 south of the fronts (Fig. 1 C).N 2 fixation rates peaked south of Madagascar and at stations near the South African coast, influenced by the Agulhas current (Fig. 1 C).N 2 fixation r ates wer e otherwise below the detection limit at most stations south of the fronts as well as at stations on the eastern part of the transect ( ∼70 • E, Fig. 1 C).Primary pr oduction r ates r anged fr om 93 to 4044 nmol C l −1 d −1 , and 182 to 2456 nmol C l −1 d −1 north and south of the fr onts, r espectiv el y (Fig. 2 B).These rates wer e mor e une v enl y distributed than N 2 fixation r ates, with the highest rates detected at stations 866 (4044 nmol C l −1 d −1 , near the South African coast, north of the fronts) and 895 (2456 nmol C l −1 d −1 , near K er guelen Islands, south of the fronts) (Fig. 1 D).While both Trichodesmium and Crocosphaera cells were found to be significantl y enric hed in 15 N north of the fronts, combining flo w c ytometry and nanoSIMS we scr eened mor e than 2,500 heter otr ophic bacteria south of the fronts and we could not find any significant enrichment (Fig. 2 ).

Diazotroph abundance
The qPCR amplification of c y anobacterial and NCDs nif H phylotypes r e v ealed substantial v ariability in both their distribution and abundance across the northern and southern regions of the fronts (Fig. 3 ).Cyanobacteria were more abundant north of the fronts, while NCDs dominated south of the fronts.Notably, our results sho w ed a co-occurrence of UCYN-A1 and Trichodesmium nif H gene counts north of the fr onts, particularl y in the Agulhas region (Fig. 3 A and B).In contrast, UCYN-A2, UCYN-B, and Gamma-A displayed a more uniform distribution across the entire sampled ar ea, irr espectiv e of the position of the fr onts (Figs 3 C-E).Furthermore, UCYN-A1 sho w ed an increased abundance under conditions c har acterized b y lo w concentrations of nitrate and phosphate (0.07-0.17 μmol l −1 and 0.02-0.09μmol l −1 , r espectiv el y).Conv ersel y, Gamma-4 was mor e abundant at stations south of the fr onts with ele v ated nitr ate (21.50-23.45μmol l −1 ) and phosphate (1.45-1.58μmol l −1 ) concentrations (Fig. 3 F).

Diazotroph community composition
We assessed diazotrophic community composition using both nifH and nifD genes as biomarkers with 1 million reads per sample .T his dual a ppr oac h r esulted in differ ent spatial distributions of diazotr oph gr oups, particularl y when divided into NCDs and c y anobacterial diazotrophs.A total of 15,938,410 reads were retrie v ed, r esulting in 1,915 ASVs from nifH amplicons, including r epr esentativ es fr om 20 phyla, 38 classes, 68 orders, 115 families, and 146 gener a. Fr om the r ecov er ed nifH ASVs, 87% wer e NCDs and 13% were cyanobacterial diazotrophs.In waters with SST < 10 • C, the most abundant groups were betaproteobacteria from the families Burkholderiaceae and Comamonadaceae , follo w ed b y Cyanoph yceae ( Trichodesmium ), thermodesulfobacteriota (Desulfocarbo ), and gamma pr oteobacteria ( Vibrio ) (Fig. 4 A).At SST le v els between 10 • C and 25 • C Cyanophyceae were the prevailing diazotrophs, follo w ed b y gammaproteobacteria, thermodesulfobacteriota, and beta pr oteobacteria.In waters with SST > 25 • C, a community shift was observed from Pseudomonadota (proteobacterial) groups to Cyanobacteria, specifically unicellular Cyanobacteria (Candidatus Atelocyanobacterium thalassa and Crocosphaera sp.), along with Trichodesmium as the dominant diazotroph taxon (Fig. 4 A).nifD gene amplicon sequencing resulted in 7,809,286 r eads, r esulting in 2,170 ASVs from 8 phyla, 12 classes, 26 orders , 47 families , and 46 genera, comprising 87% NCDs and 13% c y anobacterial diazotrophs ( Fig. S4 , Supplemental files ).Based on nifD amplicons, beta pr oteobacteria dominated the community at SST < 10 • C, follo w ed b y gamma-and alpha pr oteobacteria (Fig. 4 B).Between 10 • C and 25 • C the c y anobacterium Trichodesmium w as detected, but the diazotroph community was primarily dominated by beta-and gamma pr oteobacteria (Fig. 4 B).Abov e 25 • C the community was dominated by cyanobacteria, including Trichodesmium , Crocosphaera , Candidatus Atelocyanobacterium , Richelia , Zehria , and Hydrocoleum .NCDs exhibited higher r elativ e pr e v alence acr oss the entir e tr ansect as assessed by nifH amplicon sequencing, while the nifD amplicon sequencing a ppr oac h onl y should a higher r elativ e pr e v alence south of the STF ( Fig. S5 , Supplemental files ).
The distinctive distribution patterns observed among ASVs and envir onmental factors acr oss v arious temper atur e r anges and location with respect to the fr onts underscor ed the temper atur ede pendent d ynamics of the diazotr ophic comm unity (Fig. 5 ).Salinity and SST exhibited a positive correlation on nifD c y anobacterial community composition ( Fig. S9 , Supplemental files ).Concurr entl y, the composition of the NCDs community displayed significant correlations with N/P ratios ( r = 0.92) ( Fig. S9 , Supplemental files ).At SST < 10 • C, particularly in the south of the fronts, the distribution of nifH ASVs was str ongl y associated with F igure 5. Redundanc y analyses showing the influence of environmental variables and N 2 fixation rates on diazotroph community composition based on (A and B) nif H genes and (C and D) nif D genes, grouped as north and south of the fronts or by temperature cluster, respectively.envir onmental par ameters, including the N/P ratio, dNi, and dZn concentr ations, driving comm unity composition (Fig 5 ).Furthermore, nifD ASVs in the same temperature range displayed associations not only with the afor ementioned v ariables but also with dF e concentrations (Fig. 5 ).Con versely, in regions characterized by higher temper atur e gr adients, specificall y to the north of the fr onts (r anging fr om 10 • C to 25 • C and > 25 • C), the distribution of ASVs a ppear ed to be associated with temper atur e and dMn concentr ation (Fig. 5 ).Additionall y, the ASVs detected at these higher temper atur es wer e k e y dri vers of the observed N 2 fixation activity (Fig. 5 ).

SIO fronts divide diazotroph communities
Our results show a clear north-south divide, with substantial N 2 fixation rates north of the fronts and mostly undetectable rates south of the fronts (Fig. 1 C).While N 2 fixation was gener all y not detected south of the fronts during our study, pr e vious studies measur ed r ates up to 1.97 nmol N l −1 d −1 during the same season in this region (Hörstmann et al. 2021 ).The SIO fronts are howe v er not impermeable, with meandering and cr oss-fr ont heat and tr acer exc hange due to bar oclinic instabilities and bathymetry patterns (Chapman et al. 2020 ).Hence, we cannot rule out that pr e vious significant N 2 fixation activity measurements in previous studies ma y ha v e been influenced by diazotr oph tr ansport acr oss the fronts and/or transfer of trace metal rich waters into the reactiv e nitr ogen poor waters of the IO subtr opical gyr e.South of the fronts, the diazotroph community was dominated by NCDs (Figs 3 and 4 ), mainly composed of alpha-, beta-, gamma pr oteobacteria, and thermodesulfobacteriota (Fig. 4 ).Alpha pr oteobacteria ar e ada pted to thriv e in cold, open-ocean environments (Verde et al. 2016 ), likely explaining their significant presence at SST levels below 10 • C in our study region (Fig. 4 , Fig. S8 , Supplemental files ).Below 10 • C, alpha pr oteobacteria wer e mostl y r epr esented by the order Rhodobacterales and Hyphomicr obiales, whic h hav e a c hemoheter otr ophic metabolism and ar e capable of sulphur oxidation and carbon reduction along with N 2 fixation (Pujalte et al. 2014 ).Thermodesulfobacteriota, known for their sulfate-reducing abilities, sho w ed a dominant presence below 10 • C ( Fig. S8 , Supplemental files ).The Southern Ocean holds significant importance in the recirculation of climate-active trace gases, including dimethyl sulfoxide and methane (Thurber et al. 2020 ).This could be linked to the important presence of methanotrophs and sulfate reducers south of fronts .Likewise , beta-and gamma pr oteobacteria wer e mostl y detected at temper atur es below 10 • C range ( Fig. S8 , Supplemental files ).
Even if NCDs were dominant south of the fronts, we could not find evidence of active N 2 fixation (Fig. 2 ).A similar predominance of NCDs together with undetectable rates have been reported fr om differ ent r egions , suggesting that NCDs ma y not contribute to N 2 fixation significantly or may not be active at all (Turk-Kubo et al. 2014, Moisander et al. 2017 ).Ho w e v er, r ecent e vidence sho w ed N 2 fixation active NCDs on suspended particles in the North Pacific subtropical gyre (Harding et al. 2022 ).N 2 fixation by NCDs may respond to different conditions than those favouring c y anobacterial diazotrophs (Turk-Kubo et al. 2022 ), particularly the availability of labile dissolved organic matter in light of the lack of the photosynthetic machinery to generate ATP for N 2 fixation (Benavides et al. 2015, Riemann et al. 2022 ).Mor eov er, N 2 fixation in NCDs is likely intermittent and responds to other favouring conditions such as the presence of low oxygen microzones (e.g. in particles) and low r eactiv e nitr ogen concentr ations (Bombar et al. 2016, Bianchi et al. 2018, Chakraborty et al. 2021 ).Hence, we cannot fully rule out that NCDs fix N 2 in the SIO when conditions are fa vourable .
The observed patterns in the diazotroph community composition and N 2 fixation rates within the studied region are closely intertwined with the pr e v ailing ecological factors .T he high salinity and ele v ated temper atur es observ ed north of the fr onts a ppeared to promote the proliferation of c y anobacterial diazotrophs, likely due to their adaptation to such conditions (Furbo Reeder et al. 2022 ).Conv ersel y, the high inorganic nutrient concentrations and high N/P ratio values south of the fronts correlated negatively with the abundance of c y anobacterial diazotrophs and N 2 fixation activity ( Fig. S9 , Supplemental files ).This confirms pr e vious findings of c y anobacterial diazotr ophs being mostl y favour ed in oligotr ophic envir onments (Zehr 2011 ).While pr e vious studies hav e focused on dFe as the main trace metal impacting c y anobacterial diazotrophs, we found strong positive correlations between dMn and SST, c y anobacterial diazotroph abundance and N 2 fixation rates (Fig. 5 , Fig. S9 , Supplemental files ), suggesting a potential synergistic effect between these factors.Higher temperatures may enhance the metabolic activity of c y anobacterial diazotrophs , while dMn a vailability could serve as an essential micronutrient for their gro wth (Bro wning et al. 2021 ).In contrast, inorganic nutrients and the trace metals dNi and dZn were statisticall y r elated to the abundance of NCDs, suggesting that these diazotr ophs may thriv e in envir onments with differ ent nutrient stoichiometry than c y anobacteria.NCDs can have various metabolic capabilities including sulfate reduction, nitrate reduction, thiosulfate oxidation, and hydrocarbon and organic matter degredation (Bentzon-Tilia et al. 2015, Turk-Kubo et al. 2022 ).Trace metals like dCo, dMn, dNi, and dZn are k e y elements that boost the sulfate redcution and hydrocarbon and organic matter degradation abilities of NCDs (Luek et al. 2017 ).Increasing dNi concentrations also have been shown to enhance cellular superoxide dismutase activities and N 2 fixation rates (Ho 2013 ).These patterns suggest that further studies examining the trace metal regulation of N 2 fixation, beyond dFe are needed.

Hotspots of diazotroph activity in the SIO
Besides the main north-south frontal divide, we found two particular hotspots of diazotrophic activity: the waters around the southern tip of Madagascar and the Agulhas Curr ent.Pr e vious studies have shown significant N 2 fixation in these regions of southwest IO (Shiozaki et al. 2014, Fernández-Castro et al. 2015, Hörstmann et al. 2021, Metzl et al. 2022, Sato et al. 2022 ).Metzl et al. ( 2022 ) reported N 2 fixation rates up to 18.26 nmol N l −1 d −1 coinciding with an inter annuall y v ariable featur e known as the "Madagascar Bloom" (Longhurst 2001 ).Similarly, modelling approaches hav e r e ported high N 2 fixation acti vity in correlation with Madagascar Bloom e v ents (Tang et al. 2019 ).This phenomenon is associated with conditions favourable for N 2 fixation including increased water column stratification, dFe availability during the r ain y season due to island runoff, as well as mesoscale circulation (Metzl et al. 2022, Raes et al. 2022 ).Ho w e v er, while the strength of the Madagascar Bloom was much lo w er during our study than in pr e vious ones where high N 2 fixation rates were reported ( Fig. S11 , Supplemental files ), we measured rates up to 13.96 nmol N l −1 d −1 ar ound Mada gascar (Fig. 2 A).The high dMn concentrations and optimal SST conditions (21.79 • C-29.03 • C) ar ound Mada gascar during our cruise could hav e potentiall y driv en these high N 2 fixation r ates.Tric hodesmium was one of the most abundant diazotrophs south of Madagascar during our study, with up to 2.5 × 10 6 nif H gene copies l −1 (Fig. 3 ), as expected fr om pr e vious studies showing high abundance of this cyanobacterium in the region (Poulton et al. 2009 , Sr ok osz andQuartl y 2013 ), despite low dFe availability (Wilson and Qiu 2008 ).Ho w ever, w e also found a concomitant high abundance of UCYN-A1 (up to 5 × 10 8 nif H gene copies l −1 ; Fig. 3 ), suggesting these diazotrophs were also contributing to the high rates measured.
The Agulhas current is characterized by dynamic processes, including eddies and upwelling e v ents, whic h can lead to the entrainment and transport of nutrient-rich waters from deeper layers to the surface (Lutjeharms et al. 2000 ).Nutrient inputs combined with favourable physical conditions such as increased sunlight availability and warmer temperatures may create a fa vourable en vironment for the growth and proliferation of diazotrophic c y anobacteria like Tric hodesmium .Isotopic nitr ate signatures in the greater Agulhas area suggest active N 2 fixation takes place here (Marshall et al. 2023 ), but not in its northern and eastern br anc hes (Sigman and Fripiat 2019 ).N 2 fixation in this r egion may be sustained by high dFe concentr ations, primaril y r esulting from the resuspension of shelf sediments and atmospheric deposition (Grand et al. 2015 ).Moreover, the western Mozambique Channel shelf contributes to a phosphorus excess ( > 0.3 μM), potentially enhancing N 2 fixation in the Agulhas current (Marshall et al. 2023 ).

Biogeochemical implications
Significant alterations in the global N 2 fixation budget (from 74 ± 7 to 223 ± 30 Tg N yr −1 ) hav e primaril y been ascribed to diazotrophy in the IO (Luo et al. 2012, Shao et al. 2023 ).The total nitr ogen input thr ough N 2 fixation in the IO subtr opical gyr e has been estimated to range from 1.26 to 2.19 Tg N yr −1 , and from 2.17 to 2.27 Tg N yr −1 in the SIO ( Supplemental files ).Together, the IO subtropical gyre and the SIO contribute more fixed N 2 to the global IO than its other sub-basins , i.e .the Arabian Sea, Bay of Bengal, Equatorial IO, and Eastern IO (Chowdhury et al. 2023 ).Still, the availability of diazotrophy data in the IO is much lo w er than in the North Atlantic and Pacific oceans .T his shortfall in N 2 fixation data and the high variability of the data available to date ma y ha v e significant biogeoc hemical implications for global nitrogen budget calculations .T he dynamic nature of N 2 fixation, as e videnced by v ariations in estimates, suggests a m ultifaceted interplay of factors.Regional disparities in nutrient availability, the distribution of diazotrophs, and the influence of ocean dynamics featur es suc h as curr ents and upwelling e v ents, ar e potential drivers behind these differences .Furthermore , the variability in N 2 fixation can pr opa gate thr ough the marine food web, impacting primary productivity and fish landings .T her efor e, gaining a compr ehensiv e understanding of N 2 fixation in the IO, especially in the less-studied regions of IO subtropical gyre and the SIO, is crucial for refining our knowledge of global nitrogen cycling and its far-r eac hing ecological consequences.

Conclusions
The SIO fronts imprinted a clear latitudinal divide in nutrients and trace metal availability, coinciding with a sharp differentiation of the diazotroph community composition and associated N 2 fixation activity in the region.North of the fronts, c y anobacterial diazotrophs dominated, likely driving the high N 2 fixation activity observ ed.Conv ersel y, south of the fr onts NCDs dominated while no significant N 2 fixation activity was observed.Our findings suggest that the presence of certain trace metals, and particularly dMn, may influence the activity and composition of the diazotr oph comm unity, while others suc h as dF e , usuall y r egarded as k e y for diazotrophy, did not seem to have an impact in the SIO diazotr oph comm unity as suggested by statistical analyses.Additionall y, our r esults highlight the potential of the nif D gene as a better descriptor of NCDs taxonomy compared to the more commonly used nif H gene. Projections of net primary production increasingly div er ge in Earth system models (Tagliabue et al. 2021 ), in part due to the parameterization of N 2 fixation under climate change, with the tropical IO being among the most uncertain regions (Bopp et al. 2022 ).Hence, focusing future N 2 fixation research in the IO is needed to constrain future net primary productivity projections.Because N 2 fixation activity differs gr eatl y between diazotroph gr oups, comm unity composition data is needed along with N 2 fixation rate measurements.Our data provides a comprehensive insight into N 2 fixation activity and diazotroph community composition in the SIO, contributing to filling this gap.

Figure 1 .
Figure 1.CTD profile and underway stations sampled during the cruise overlaid on (A) sea surface temperature (SST) and Chlorophyll-a composite of L3M 4 km product retrieved from the Copernicus marine service for the cruise period (13 January 2021 to 4 March 2021).STF = subtropical front, SAF = subantarctic front, and PF = polar front.(C) N 2 fixation (nmol N l −1 d −1 ) and (D) primary production (nmol C l −1 d −1 ) rates measured from surface samples (5 m) along the cruise transect.Nondetectable rates are depicted with crosses.AC = Agulhas current and ARC = Agulhas return current.

Figure 2 .
Figure 2. Whisker plot of 15 N cellular fractional abundance (atom%) for eac h gr oup anal yzed.Eac h dot r epr esents a single anal yzed cell.Gr ay dots denote cells with rates not significantly different from zero.Black lines denote mean 15 N fractional abundance and standard deviations (horizontal plain and vertical dashed, respectively). 15N enrichments of heter otr ophic bacteria were significantly different from Crocosphaera and Tric hodesmium (Wilk oxon test, P < .001),but not Crocosphaera and Tric hodesmium 15 N enric hments wer e not significantl y differ ent fr om each other (Wilkoxon test, P = .92).

Figure 4 .
Figure 4. Heatma p r epr esenting most abundant ASVs acr oss thr ee temper atur e gr adients for the (A) nif H gene , and (B) nifD gene .nif H groups ha ve been r eorder ed to facilitate comparison with nif D gr oups.
While c y anobacteria dominated north of the fronts, we highlight the detection of Trichodesmium south of the fronts (Fig.4).The presence of Trichodesmium on both sides of the fronts may r esult fr om adv ection, as observ ed for other organisms with positive buo y anc y(F raser et al. 2022 ).Alternatively, the presence of Trichodesmium south of the fronts may be explained by their polew ar d migration, indicating potential adaptation to colder waters.While capable of growth at temperatures below 20 • C (Rivero-Calle et al. 2016 ), Trichodesmium may experience compromised N 2 fixing efficiency under such conditions.Numerical advection modelling indicates that Trichodesmium communities can endure for over 3.5 months at temper atur es below pr e vious expectations(Rees et al. 2016 ), suggesting their occupancy of a distinct nic he r elativ e to other Trichodesmium species as a cold-or low-light-ada pted v ariant from the SIO.